1. Field of the Invention
The present invention relates to a method of determining the site-specificity for the binding of an analyte to a ligand having more than one binding site, as well as to a kit for carrying out the method. The invention also relates to an analytical system, a computer program product and a computer system for performing the method.
2. Description of the Prior Art
An important function of blood proteins is to permit reversible binding of ligands, such as fatty acids, hormones, amino acids, cations, etc, to ensure that they are distributed throughout the body. Therefore, when a drug is administered to a living body it is usually bound to a smaller or larger extent to blood (plasma) proteins. The unbound drug concentration in plasma is therefore being more closely related to the pharmaceutical activity than the total concentration of drug. The major protein in plasma responsible for binding of many kinds of drugs is human serum albumin (HSA). There are at least six different binding sites spread over the HSA molecule and to which drug molecules may bind. Determining the binding propensity of a drug candidate to HSA is therefore essential in evaluating drug candidates. Traditionally, binding properties have been examined using techniques such as equilibrium dialysis, fluorescence, circular dichroism, column chromatography, or titration calometry. Recently, also surface plasmon resonance (SPR) biosensors have been used (Frostell-Karlsson, Å, et al., J. Med. Chem. 43, 1986-1992; Rich, R., et al., Anal. Biochem., 296, 197-207).
In addition to the binding parameters, it is also important to know to which specific binding site on the HSA molecule that the drug candidate binds. For example, co-administration of drugs which share the same binding site can cause an increase in the unbound drug concentrations due to binding competition, and may result in undesirable side effects or unexpected changes in drug disposition. To identify the binding site, X-ray crystallography may be used. Alternatively, site-specificity may be determined by studying competition with substances whose site specificities are already known, using techniques, such as, for example, circular dichroism, microcalometry or affinity blotting. While no such determination of a drug candidate's HSA-binding site by competitive SPR biosensor techniques seems to have been reported in the literature, the use of SPR biosensor technology may, of course, also be contemplated.
Naturally, there are numerous other ligands than HSA that have more than one binding site and for which it may be of interest to determine the site-specificity of drug candidates and other binding species.
U.S. Pat. No. 5,753,518 discloses the use of mass-sensing methods, especially SPR biosensor technology, to determine the binding of low molecular weight analytes, such as drugs, to a common receptor (ligand) immobilized to a sensing surface. The molecular weights of the analytes are often considered to be too low to give detectable responses when binding to the sensing surface. Each low molecular weight analyte is therefore mixed with an analyte analogue having a substantially higher molecular weight than the low molecular weight analyte, the mixture is contacted with the surface, and the response of each mixture is compared with that of the analyte analogue alone. The distorsion of the analyte analogue response (lowering of the response level) is representative of the affinity and kinetic properties of the low molecular weight analyte. While the response usually is in the form of a binding curve (response vs. time), it is not necessary to study the response up to steady state or equilibrium but the initial part of the association phase is sufficient.
It is an object of the present invention to provide an improved method of determining the binding site specificities of analytes to a ligand having multiple binding sites.